More complicated Compounds have been developed in the field of pharmacology and agricultural chemistry, and there have been an increasing number of compounds having more than one asymmetric carbon. There are commercially available compounds that have been on the market as a racemic mixture. In many cases, it has been reported that only one optically active compound in the racemic mixture exhibits biological activities, and the other compounds exhibit no biological activity, or are harmful to mammalians or environments. Accordingly, it is necessary to develop biological and/or chemical techniques that can synthesize a desired single isomer. As the biological and/or chemical techniques that can synthesize only one optically active compound, an asymmetric synthesis, a method for concentrating an enantiomer, or stereospecific synthesis of an organic compound using enzymes, etc. have been recently studied all over the world, and, among them, many studies on the optical resolution of a racemic mixture using amidase has been reported.
Esfenvalerate [(S)-α-cyano-3-phenoxybenzyl (S)-2-(4-chlorophenyl)-3-methylbutyrate] whose much attention has been recently attracted in the racemic mixture is a chiral isomer of racemic fenvalerate, a biological activity of which results mainly from (S)-isomers (FIG. 1a).
(S)-2-(4-chlorophenyl)-3-methylbutyric acid used for the synthesis of esfenvalerate may be synthesized using various methods. As one of the methods for producing an optically active compound (R)— or (S)—(S)-2-(4-chlorophenyl)-3-methylbutyric acid, there is an optical resolution technique in which optically pure amine (for example, (R)- or (S)-phenylethylamine) as a resolving agent is added to a racemic mixture and a desired compound is separated from the racemic mixture. However, the problem is that the resolving agent is very expensive and its process is complicated.
Also, as one of the methods for synthesizing (S)-2-(4-chlorophenyl)-3-methylbutyric acid, there is a method employing lipase or esterase by using ester as a starting material, or a method employing nitrile hydratase having stereoisomeric selectivity (Fallon et al., Appl. Microbiol. Biotechnol. 47: 156, 1997).
Meanwhile, in order to optically resolve a certain racemic mixture, methods for selectively hydrolyzing an enantiomer using an enzyme such as esterase, amidase (lipase) and protease has been known. For example, a method for hydrolyzing a Candida rugosa-derived lipase using racemic methyl-2-chloropropionate(Methyl-2-chloropropionate) has been known (Dahod & Siuta-Mangano, Biotech. Bioeng., 30:995, 1987). Also, a method for synthesizing (R)-2-(4-hydroxyphenoxy)propionic acid using a purified Candida rugosa-derived amidase has been reported (WO 1990/15146).
It is very effective to use an enzyme for the optical resolution of a racemic mixture, but it is not only very difficult to confirm which racemic compounds are optically resolved with enzyme, but also to confirm which enzymes are particularly effective to resolve racemic compounds. For example, U.S. Pat. No. 5,928,933 discloses that, in order to optically resolve racemic 4-oxo-1,2-pyrrolidinedicarboxylic acid dialkyl ester, 44 enzymes selected from proteases, amidases and esterases was tested for specificity of enzyme activity, and one of the 44 enzymes shows an optical purity of 95%. As described above, it is very important to find suitable combinations of enzymes with substrates through continuous studies since the selectivity and optical purity (% ee) of isomers are varied according to the kinds of the used enzymes and the chemical structure of the substrate, etc.
Meanwhile, in the case of the racemic (R),(S)-2-(4-chlorophenyl)-3-methylbutyramide which is the subject for the optical resolution in the present invention, it is yet not known that the racemic mixture is optically resolved using amidase. The conventional optical resolution using enzymes is mainly limited to the synthesis of an intermediate, aryloxypropionic acid, of a prop-based herbicide, the synthesis of an intermediate, arylpropionic acid, of a profen-based antiinflammatory agent, etc., but there is no precedent for using enzymes for the optical resolution of (R),(S)-2-(4-chlorophenyl)-3-methylbutyramide.
Meanwhile, the amidase, which hydrolyzes carboxylic acid amide, belongs to Enzyme Class E.C.3.4. It has been known that the amidase, reported until now, is extensively present in the microorganism such as Corynebacterium, Pseudomonas, Bacillus, Brevibacterium, Rhodococcus, Alcaligenes, etc. It has been known that the amidases are mainly induced and expressed to exhibit specific substrate specificities in every microorganism (Martinkova & Kren, Biocat. Biotrans., 20:73, 2002). In particular, Pseudomonas putida ATCC 12633-derived amidase is used in the method used by DSM (the Netherlands), which is a method for synthesizing D-amino acid or L-amino acid using D,L-amino acid amide (Sonke et al., Stereoselective Biocatalysis, p 23-58, 2000, Patel, R. N. ed., Marcel Dekker). For example, the Pseudomonas putida ATCC 12633-derived amidase has been reported as a useful enzyme that stereoselectively hydrolyze L-phenylglycinamide into D-phenylglycinamide and L-phenylglycine (Hermes et al., Appl. Environ. Microbiol., 59:4330, 1993). However, its substrate specificity is not completely characterized, and there is a need for a novel amidase which may be produced more economically and react with substrates that are not converted by the conventional amidase.
Accordingly, there are urgent needs for screening and developing a novel amidase capable of being used for the optical resolution, a novel strain capable of producing the amidase, and a method for optically resolving a racemic mixture using the novel strain in the art.